Tim Hunkapiller

Mouse T cell antigen receptor: Structure and organization of constant and joining gene segments encoding the β polypeptide

The germ-line joining (J) gene segments and constant (C) genes encoding the beta chain of the mouse T cell antigen receptor have been isolated on a single cosmid clone. There are two constant genes, C beta 1 and C beta 2, each associated with a cluster of J beta gene segments. The nucleotide sequences of the C beta 2 gene and of the J beta 2 cluster gene segments have been determined. The coding sequence of the C beta 2 gene is very similar to the sequence of a cDNA clone encoded by the C beta 1 gene. The C beta 2 gene has four exons; exon-intron structure does not obviously correspond to the functional domains of the protein. The J beta 2 gene segment cluster contains six functional J gene segments. We have isolated specific probes for the C beta 1, C beta 2, J beta 1, and J beta 2 regions to examine DNA rearrangements in T lymphocytes. DNA rearrangements can occur in both J beta gene segment clusters, and both C beta genes appear functional.

Diversity of the immunoglobulin gene superfamily.

Publisher Summary The physical nature and organization of particular genetic information establish the limits and possibilities of its variation. The multigene organization and recombinogenic nature of much of the family establishes particular diversifying potentialities on many of the members. New functional possibilities arise primarily through the duplication of the various informational units involved, for example, nucleotides, exons, genes, and entire multigene families. Duplication of a multigene family and the attendant cis-acting control mechanisms can in a single event create the genetic basis of a complex new phenotype, suggesting the possibility of rapid evolutionary change. Interestingly, the number of the identified single-gene IgGSF members that are expressed in both the brain and the immune system suggests the possibility of shared cell surface recognition functions and that the related molecules may be involved in some of the intriguing phenomena linking the mental states and immune response. It seems likely that the fruitful strategies of somatic diversification, employed by well-characterized members of the IgGSF, will also be used by other receptor families, perhaps involved in the morphogenesis or neuronal development.

The murine T-cell receptor uses a limited repertoire of expressed V|[beta]| gene segments

Only 10 different Vβ gene segments were found when the sequences of 15 variable (Vβ) genes of the mouse T-cell receptor were examined. From this analysis we calculate that the total number of expressed Vβ gene segments may be 21 or fewer, which makes the expressed germline Vβ repertoire much smaller than that of the immunoglobulin heavy-chain or light-chain genes. We suggest that βchain somatic diversification is concentrated at the Vβ−Dβ−Jβ junctions.

Three cDNA clones encoding mouse transplantation antigens: Homology to immunoglobulin genes

We constructed cDNA libraries from poly(A)+ RNA isolated from cell lines of two different inbred strains of mice, and screened the libraries with a cDNA clone encoding a human transplantation antigen. Three cDNA clones were identified, sequenced and found to encode amino acid sequences highly homologous to portions of a known mouse transplantation antigen. Comparison of the cDNA sequences of mouse transplantation antigens with the constant region domains of the mouse immunoglobulin mu gene reveals a striking homology, which suggests that the two genes share a common ancestor. Antibody genes undergo DNA rearrangement during B cell differentiation that are correlated with their expression. In contrast, DNA blots with these cDNA probes suggest that the genes for the transplantation antigens are not rearranged in the genomes of liver or embryo cells, which express these antigens, as compared with sperm cells, which do not express these antigens. In Bam Hl-digested liver DNAs from different inbred strains of mice, 10-15 bands of hybridization were found. Accordingly, the genes encoding the transplantation antigens appear to constitute a multigene family with similar gene numbers in different mice.

Chimeric immunoglobulin-T cell receptor proteins form functional receptors: Implications for T cell receptor complex formation and activation

We constructed chimeric receptor chains in which an immunoglobulin heavy chain variable region (VH) from a phosphorylcholine-specific antibody is substituted for T cell receptor (Tcr) alpha and beta V regions. We demonstrate that the VH region joined to either the C alpha or the C beta region can form stable chimeric proteins in EL4 T cells. Both chimeric receptor chains associate with CD3 polypeptides in functional receptor complexes and respond to phosphorylcholine coupled to Sepharose beads. The VH-C alpha chimeric chain associates with the EL4 beta chain, while the VH-C beta chimeric protein appears to form either a homodimer or a heterodimer with the native EL4 beta chain. Thus, functional receptor complexes can be formed using two C beta regions, and the C alpha region may not be required for CD3 association and surface expression of Tcr complexes.

Rearranged β t cell receptor genes in a helper t cell clone specific for lysozyme: No correlation between Vβ and MHC restriction

Abstract The helper T cell clone 3H.25 is specific for hen egg white lysozyme and the class II MHC molecule I-A b . This T H cell has three rearrangements in the β-chain gene family-a V β -D β -Jp β1 and a D β2 -J β2 rearrangement on one homolog and a D β1 -J β2 rearrangement on the other. These observations demonstrate that this functional T lymphocyte expresses only a single V β gene segment and, accordingly, exhibits allelic exclusion of β-chain gene expression. The rearranged 3H.25 V β gene segment is the same as that expressed in a T helper cell specific for cytochrome c and an I-E k MHC molecule. Thus, there is no simple correlation between the V β gene segment and antigen specificity or MHC restriction.

The T cell differentiation antigen Leu-2/T8 is homologous to immunoglobulin and T cell receptor variable regions

Leu-2/T8 is a cell surface glycoprotein expressed by most cytotoxic and suppressor T lymphocytes. Its expression on T cells correlates best with recognition of class I major histocompatibility complex antigens, and it has been postulated to be a receptor for these proteins. We have determined the complete primary structure of Leu-2/T8 from the nucleotide sequence of its cDNA. The protein contains a classical signal peptide, two external domains, a hydrophobic transmembrane region, and a cytoplasmic tail. The N-terminal domain of the protein has striking homology to variable regions of immunoglobulins and the T cell receptor. The membrane-proximal domain appears to be a hinge-like region similar to that of immunoglobulin heavy chains. The superfamily of immunologically important surface molecules can now be extended to include Leu-2/T8.

Biological information signal processor

The computation requirements for mapping and sequencing the human genome might soon exceed the capability of any existing supercomputer. The systolic array processor presented in this paper, called biological information signal processor (BISP), has the capability to satisfy the current and anticipated future computational requirements for performing sequence comparisons based on the T.F. Smith and M.S. Waterman algorithm (1981) as extended by M.S. Waterman and M. Eggert (1987). The BISP can conduct the most time consuming sequence comparison functions, establishing both global and local relationships between two sequences. A modified Smith and Waterman algorithm is presented in this paper for efficient VLSI implementation. Methods are developed to reduce the BISP systolic array I/O bandwidth problem by reporting only the statistical significant results. Estimated performance of the BISP is compared with several different computer architectures.<<ETX>>

An automated DNA synthesizer employing deoxynucleoside 3'-phosphoramidites.

Publisher Summary This chapter describes the design of an automated deoxyribo nucleic acid (DNA) synthesizer that can synthesize longer strands of DNA at lower cost. Moreover, two, three, or four bases can be incorporated at appropriate positions in accordance with the ambiguity of the genetic code for individual amino acid residues. The design and operation of such a synthesizer is described in this chapter. The primary reactor is a flow through column, and the reagent and solvent deliveries are controlled by a series of zero-dead volume, pneumatically actuated diaphragm valves. To move reagents and solvents from the reservoirs, positive argon pressure is used rather than a mechanical pump. This chapter has carried out extensive studies, concerning the synthesis of DNA mixtures. This chapter discusses the using color-coded 5’ protecting groups on each of the four bases and computer analysis for multiple absorption spectrum analysis. This chapter discusses the ability to prove the equimolar existence of all desired sequences in the final mixture.

Large-scale DNA sequencing

Abstract The problems of large-scale sequencing are discussed, together with existing and future strategies, and the technology capable of achieving the goal of a productive, efficient process is reviewed.